1. Field of the Invention
The present invention relates to a recombinant soluble human thyroid hormone receptor (hereinafter referred to as “sTSHR”) which is secretory and has reactivity with an anti-human thyroid stimulating hormone receptor autoantibody; a process for producing sTSHR, comprising infecting an insect cell, particularly Hi five cell, with a recombinant baculovirus prepared by inserting a gene encoding sTSHR, and culturing the infected cells; a reagent for assaying an anti-human thyroid stimulating hormone receptor antibody, such as an autoantibody, using sTSHR; and a method for measuring an anti-human thyroid stimulating hormone receptor antibody, such as an autoantibody, using sTSHR.
2. Brief Description of the Background Art
A human thyroid stimulating hormone receptor (hereinafter referred to as “TSHR”) is a receptor of thyroid stimulating hormone (hereinafter referred to as “TSH”) which is present on the thyroid membrane. When TSH secreted from the pituitary gland binds to TSHR on the thyroid follicle cell membrane, the thyroid gland secretes T3 and T4 having metabolic functions. TSHR is a seven transmembrane receptor having a molecular weight of about 95,000 to 100,000.
Graves' disease is a hyperthyroidism induced by the acceleration of formation and secretion of thyroid hormones. As its cause, the presence of a stimulative substance which quickens secretion of thyroid hormones in patient's serum can be enumerated. It is known from the studies until now that an autoantibody for TSHR is present in patient's serum and induces hyperthyroidism by activating a thyroid stimulating hormone receptor. Thus, the measurement of the autoantibody for TSHR has a considerable significance in carrying out clinical diagnosis.
The measurement of an anti-TSHR autoantibody has so far been carried out by the method developed by Smith (Endocr. Rev., 9: 106-120 (1988)). In this method, the anti-TSHR autoantibody is measured by using a porcine thyroid gland membrane fraction as the TSHR source and by allowing 125I-labeled bovine TSH and an anti-TSHR autoantibody in patient's serum to compete with each other for the TSHR source.
However, since a cross reaction, namely binding of porcine TSHR to an anti-human TSHR autoantibody in human serum, is examined in the conventional method, there is a possibility that the assay result does not correctly reflect binding of human TSHR originally formed in the living body to the anti-human TSHR autoantibody in human serum. Also, since sequences of amino acid residues of human TSHR and porcine TSHR are actually different from each other, it is expected that the results of the conventional method do not coincide with the binding of the human TSHR autoantibody to the human TSHR. In addition to these problems, there is another problem in that it is difficult to prepare the porcine thyroid gland membrane fraction used as the TSHR source at a large amount.
Naturally, it is preferred to use human TSHR for the measurement of an autoantibody for human TSHR. However, since it is impossible in reality to obtain natural TSHR from human, attempts have been made to prepare it by genetic recombination techniques. Particularly, in order to purify TSHR by expressing it at a large amount, it is important to create TSHR which has reactivity with anti-human TSHR antibody and is secretory.
TSHR is a seven transmembrane receptor and its first N-terminus extracellular domain occupies the majority of TSHR, so that it is considered that the binding region for an anti-human TSHR autoantibody is present in this region. Although attempts have so far been made by a plurality of research groups to express soluble TSHR constituted by the first N-terminus extracellular domain of TSHR at a large amount using insect cells or animal cells, the expressed soluble TSHR is accumulated as an insoluble protein inside the cells in each case, without success in effecting extracellular secretion and purifying a large amount of the soluble TSHR (Journal of Molecular Endocrinology, 10: 127-142 (1993)); Endocrinology, 138: 1658-1666 (1997); The Journal of Biological Chemistry, 270: 1543-1549 (1995); Journal of Immunology, 158; 2798-2804 (1997); Molecular and Cellular Endocrinology, 147: 133-142 (1999); Endocrinology, 138: 1559-1566 (1997); Autoimmunity, 14: 315-320 (1993)). In addition, it has been reported that the soluble TSHR does not have affinity for TSH and shows only a weak reactivity for an anti-TSHR antibody existing in serum from patients with Graves' disease.
It has been reported that a soluble TSHR (aal-309) in which 106 amino acid residues were deleted from the extracellular domain C-terminus of TSHR was secreted into extracellular moiety in CHO cells (The Journal of Biological Chemistry, 272: 18959-18965 (1997)). However, this C-terminus deleted soluble TSHR does not have affinity for TSH, and it is considered that epitope of an anti-TSHR antibody derived from patients with Graves' disease is also present in the deleted region, so that it cannot be used in the measurement of anti-TSHR autoantibodies.